Dietary or pharmaceutical composition for use for the prevention or treatment of hyperoxaluria

ABSTRACT

This invention provides materials and procedures for the delivery of selected strains of bacteria and/or oxalate-degrading enzymes to the intestinal tracts of persons who are at increased risk for oxalate related disease because they have lost, or have inadequate concentrations of these bacteria. The administration of these bacteria and/or the relevant enzyme removes oxalate from the intestinal tract and thus reduces the amount of oxalate available for absorption and reduces the risk for oxalate related disease.

This application is a continuation of Application No. PCT/IT00/00213,filed May 24, 1999 the entire content of which is hereby incorporated byreference in this application.

The present invention relates to the use of bacterial species and/orstrains that will be indicated later, for preparing a composition forthe prevention and/or treatment of hyperoxaluria and of disordersassociated with this, as well as the composition thus obtained.

Correspondingly, the said composition can assume the form and performthe activity of a dietary composition or of a food supplement or of areal drug, depending on the supporting or preventive or trulytherapeutic action that the composition is intended to exert dependingon the particular individuals for whom it is intended. This preventiveor truly therapeutic action can derive from colonization, by the saidbacteria, of the intestine of subjects at risk of hyperoxaluria ordisorders associated with hyperoxaluria.

Hyperoxaluria consists of excessive presence of oxalates in the urine(urinary oxalate >40 mg/die). As well as being caused by a geneticdefect that alters the metabolism of glyoxylic acid with formation ofoxalate instead of glycine, it may be a side effect of excessiveingestion of foods rich in oxalate, such as spinach, cocoa, hazelnuts,pepper and tea, or treatment with, for example, anti-obesity drugs (e.g.ORLISTAT). Hence the usefulness of providing a dietary supplementcapable of reducing and regulating the presence of oxalates in theurine.

Hyperoxaluria is a predominantrisk factor for the formation of renalcalculi an may be caused by excessive absorption of oxalate from thecolon or by renal overload caused by hyperoxaluria as in primaryhyperoxaluria (PH).

The urinary level of oxalate seems to constitute a crucial sign withregard to the formation of calcium oxalate calculi even in patients withnormal urinary excretion of calcium, and calcium oxalate calculi cansometimes also form in patients under strictly oliguric dialysis andwithout a prior history of nephrolithic disease in that, within thisframework, there may be oxalate supersaturation even in a minimalquantity of urine.

The daily excretion of oxalate is related to urinary volume, to thetaking of vitamin C, to the body weight index and, inversely, to theintake of calcium.

A study by Sutton and Walker on the population of idiopathic calciumoxalate calculi formers with slight hyperoxaluria was unable todemonstrate any significant alteration of renal control of oxalate, andconcluded there was an increased dietary burden of oxalate with apossible hyper-absorption mechanism.

In non-PH hyperoxaluria patients it will therefore be necessary to paygreatest attention to enteral hyperabsorption of oxalate, which promotesthe clinical picture of enteric hyperoxaluria and often the recurrentproduction of renal calculi. It has in fact been recognized since 1968that nephrolithiasis is a complication of a disease or of resection ofthe intestine.

Increased intestinal absorption of oxalate, termed enteric hyperoxaluria(EHO), depends on at least two mechanisms. The first is associated withmalabsorption of biliary salts in the diseased or resected ileum, whichcauses a deficiency of biliary salts and malabsorption of fats. Most ofthe oxalate in the diet is bound to calcium and is securely absorbed,but poorly absorbed fats bind intraluminal calcium, lowering thequantity bound to oxalate and giving rise to increased absorption ofoxalate. The second mechanism of EHO is associated with increasedpermeability of the colon for oxalate, caused by poorly absorbed fattyacids and biliary salts, perhaps aggravated by variations of theepithelial occluding junctions of the colon caused by the decrease inintralumial calcium. This hyperoxaluria is related to the degree ofsteatorrhoea, and is unusual with ileal resections <30 cm. A decreasedcount of bacteria that metabolize the colon oxalate (Oxalobacterformigenes) as well as their inhibition by poorly absorbed biliary saltscan be likewise contribute to EHO. Finally, hyperoxaluria can beobserved with long-term parenteral nutrition, also in patients withcolectomies and minimal intake by the oral route, perhaps because ofincreased synthesis of endogenous oxalate.

Oxalate is sparingly soluble in water, but the urine can becomesupersaturated through the presence of crystallization inhibitors.Hyperoxaluria, combined with a decreased volume of urine and reducedlevels of these inhibitors, predisposes to renal calculi.

Parenchymal renal deposition of oxalate can cause interstitial nephritisand nephrocalcinosis, with acute or chronic renal insufficiency.

The treatment of patients with calcium oxalate calculi is complex, andis described below.

Stage 1

Increased intake of liquids for a urinary excretion of 3 l/day;

Diet low in oxalate (avoid spinach, rhubarb, beets, hazelnuts, tea,cola, chocolate, wheat bran, strawberries);

Low-fat diet (50 g/day);

Calcium supplement (1-2 day);

Cholestyramine (4 g four times a day);

Stage 2 (if Calculi Recur Despite the Treatment of Stage 1)

Alkalization of the urine and citrate supplement (for example potassiumcitrate, sodium citrate=30 mEq base four times a day);

Magnesium supplement (to correct the urinary levels);

Allopurinol 300 mg/day (if the calculi contain uric acid).

The possibility of gastroenteric biological manipulation of oxalate hadbeen known since 1955 from documents on the destruction of oxalate bythe contents of the cow rumen and, subsequently, by mixed bacterialflora of the large intestine of other herbivores.

Allison referred in 1985 to the specific effect of degradation ofoxalate of Oxalobacter formigenes, which inhabits the large intestine ofman as well as of other animals.

More recently, Ito demonstrated the degradation of the oxalate contentof foods in vitro by means of Eubacterium lentum EIH-1.

Subsequently, the absence of Oxalobacter formigenes was suggested as arisk factor for hyperoxaluria in cystic fibrosis patients.

Enteric hyperoxaluria is therefore the result of excessive entericabsorption of oxalate through increased permeability of the mucosa orincreased solubility and bioavailability of faecal oxalate such as whenthe calcium content of the diet is reduced. The absence of Oxalobacterformigenes can add a new pathophysiological mechanism that is importantfrom the standpoint of therapy.

Now it has been found, surprisingly, that the following bacteria:Streptococcus thermophilus, Lactobacillus brevis, Lactobacillusacidophilus, Lactobacillus plantarum, Bifidobacterium infantis,Bifidobacterium longum and Bifidobacterium breve are capable of growingin the presence of and/or of degrading oxalate.

Accordingly, the present invention provides the use of at least onestrain of the following bacteria: Streptococcus thermophilus,Lactobacillus brevis, Lactobacillus acidophilus, Lactobacillusplantarum, Bifidobacterium infantis, Bifidobacterium longum andBifidobacterium breve for preparing a dietary and/or pharmaceuticalcomposition for the prevention and/or treatment of hyperoxaluria and thedisorders associated with this.

Preferably, the strain of Lactobacillus brevis is the strain ofLactobacillus brevis CD2 deposited at the DSM—Deutsche Sammlung vonMikroorganismen und Zelkulturen GmbH, Braunschweig, Germany, on Feb. 6,1998 with accession number DSM 11988 under the Budpest Treaty, ormutants and derivatives thereof.

More particularly, hyperoxaluria and the disorders associated with itcomprise enteric hyperoxaluria, renal calcium oxalate lithiasis,hyperoxalurias from intestinal inflammatory diseases, renalinsufficiency, vesical calculosis, cardiopathy from hyperoxaluria,cystic fibrosis and vulvodynia.

Use of the invention includes use in the veterinary field.

According to the invention, the dietary and/or pharmaceuticalcomposition is able to colonize, with the said bacteria, the intestineof subjects at risk of hyperoxaluria or disorders from hyperoxaluria, ordisorders from renal calcium oxalate lithiasis.

The invention likewise provides a dietary or pharmaceutical compositioncomprising at least one strain of the following bacteria: Streptococcusthermophilus, Lactobacillus brevis, Lactobacillus acidophilus,Lactobacillus plantarum, Bifidobacterium infantis, Bifidobacteriumlongum and Bifidobacterium breve.

According to one embodiment of the invention, a strain of Streptococcusthermophilus is combined with a strain of Lactobacilius selected fromthe group consisting of Lactobacillus brevis, Lactobacillus acidophilusand Lactobacillus plantarum or their mixtures. Preferably, the strain ofLactobacillus brevis is the strain of Lactobacillus brevis CD2 depositedat the DSM—Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH,Braunschweig, Germany, on Feb. 6, 1998 with accession number DSM 11988under the Budapest Treaty, or mutants and derivatives thereof.

According to another embodiment of the invention, a strain ofStreptococcus thermophilus is combined with a strain of Bifidobacteriumselected from the group consisting of Bifidobacterium infantis,Bifidobacterium longum and Bifidobacterium breve or their mixtures. Inthis case, preferably, the proportions between the concentrations ofBifidobacterium infantis, Bifidobacterium longum and Bifidobacteriumbreve in the mixture, expressed in CFU/g of composition, are preferably1:1:1.

According to a preferred embodiment, the ratio between theconcentrations of Streptococcus thermophilus and of the bacterium of thegenus Lactobacillus, expressed in CFU/g of composition, is from 1000:1to 1:1000, whereas the ratio between the concentrations of Streptococcusthermophilus and of the bacterium of the genus Bifidobacterium or themixture of bacteria of the genus Bifidobacterium, expressed in CFU/g ofcomposition, is from 1000:1 to 1:1000.

The total concentration of the bacteria is preferably from 10⁶ to 10¹²CFU/g of composition.

According to the invention, the composition can moreover contain or beadministered in combination with:

-   -   substances capable of binding the oxalate in the intestinal        lumen, in particular cholestyramine (for example 0.5-4 g/die)        and organic hydrocolloids of marine origin,    -   vitamins, in particular B₆ (for example 20-200 mg/die) and C        (for example 0.1-2 g/die),

magnesium oxide (for example 50-600 mg/die),

calcium (for example 0.5-2 g/die),

allopurinol (for example 50-300 mg/die),

-   -   enzymes, lactic bacteria, hormones and diuretics,        immunomodulators, anticancer, lipids, urine alkalizers, urine        acidifiers, saturated and unsaturated fatty acids and        phospholipids.    -   drugs bringing about hyperoxaluria as a side effect, for example        anti-obesity drugs (e.g. ORLISTAT).

Generally, the composition can be administered orally or by theintraluminal route or by enteroclysis in the form of granules, tablets,capsules, suppositories or by enteroclysm.

The invention also provides a food based on chocolate, cocoa, asparagus,tomato, drinks/liquids, spinach, walnuts, hazelnuts, fibres, cereals,potato, tea and peanut butter, containing a quantity of at least onestrain of bacteria selected from among Streptococcus thermophilus,Lactobacillus brevis, Lactobacillus acidophilus, Lactobacillusplantarum, Bifidobacterium infantis, Bifidobacterium longum andBifidobacterium breve, sufficient to colonize the intestine of subjectsat risk of hyperoxaluria or disorders arising from hyperoxaluria.

The invention finally provides a method for the prevention and/ortreatment of hyperoxaluria and the disorders associated with this byadministering, to a subject at risk of hyperoxaluria or a disorderarising from hyperoxaluria, from 0.5 to 4 g/die of the composition ofany one of the claims from 4 to 10.

“Subjects” in this context includes humans and animals in general, andin particular farm animals, sport animals and pets.

EXAMPLE 1

Pure Cultures

Bifidobacterium infantis, Lactobacillus acidophilus, Lactobacillusplantarum, Lactobacillus brevisand Streptococcus thermophilus wereemployed for the following experiment.

All the strains were stored lyophilized in a refrigerated environment.Bifidobacterium infantis was cultivated in MRS broth (DIFCO)+0.5%glucose, incubated anaerobically at 37° C. for 18 hours in Gas Pak withatmosphere of CO₂ and H₂, the Lactobacillus acidophilus andLactobacillus plantarum in MRS (DIFCO) broth, incubated at 37° C. for 18hours, the Lactobacillus brevis in MRS (DIFCO) broth, incubated at 30°C. for 18 hours, and the Streptococcus thermophilus in MI7 (DIFCO)broth+0.5% of lactose and incubated at 37° C. for 18 hours.

Media

For preparation of the culture media containing ammonium oxalate (BDH)10 mM and 20 mM, to 10 ml of base medium (formulated as follows: 10 g ofproteoses peptone No. 3 (DIFCO)+5 g of yeast extract (DIFCO)+1 ml ofTween '80 (DIFCO)+2 g of KH₂PO₄ (BDH)+5 g of sodium acetate (BDH)+2 g ofammonium dihydrogencitrate (MERCK)+0.05 g of MgSO₄.7H₂O (MERCK)+0.05 gof MnSO₄ (MERCK)+water qs. to 500 ml and sterilized at 121° C. for 15min) 10 ml of the following sugars and ammonium oxalate solutions,sterilized with 0.45 μm filter, were added, and namely:

for L. acidophilus, L. plantarum and L. brevis

1A: ammonium oxalate 20 mM+40 g/l of glucose (BDH)

1B: ammonium oxalate 40 mM+40 g/l of glucose for S. thermophilus

2A: ammonium oxalate 20 mM+40 g/l of glucose+10 g/l of lactose (DIFCO)

2B: ammonium oxalate 40 mM+40 g/l of glucose+10 g/l of lactose

for B. infantis

3A: ammonium oxalate 20 mM+50 g/l of glucose

3B: ammonium oxalate 40 mM+50 g/l of glucose

After mixing, the samples labelled A contain ammonium oxalate 10 mM,whereas the samples labelled B contain ammonium oxalate 20 mM.

The culture broths were then inoculated at 10% with a fresh culture andincubated in the appropriate conditions of each strain described above.

Each grown broth culture was submitted to counting of the number ofmicroorganisms contained in the culture media in the appropriateconditions, and in particular:

B. infantis in agar HHD (HI Media Laboratories), incubated anaerobicallyat 37° C. for 3 days;

L. acidophilus in agar MRS (DIFCO), incubated anaerobically at 37° C.for 3 days;

L. plantarum and L. brevis in agar MRS (DIFCO), incubated anaerobicallyat 37° C. for 3 days;

S. thermophilus in agar MI7 (DIFCO), incubated at 37° C. for 2 days.

Determination of Oxalic Acid

Sample Preparation

Broth cultures were pasteurized at 90° C. for 15 min, then centrifugedat 5000 rpm for 10 min, and finally the supernatant was filtered with a0.45 μm filter.

Method

The oxalic acid content was determined with the “Oxalic acid” kit(Boehringer Mannheim) specific for this acid. The analysis was performedwith a spectrophotometer (Perkin Elmer—Lambda 5) at 340 nm.

Results

As can be seen from Table 1 below, all the strains generally develop inthe presence of ammonium oxalate 10 mM, whereas a concentration of 20 mMpartly inhibits microbial growth, especially in the case of L.acidophilus and S. thermophilus.

However, there does not seem to be a relation between bacterialdevelopment and degradation of the oxalate. Thus, L. plantarum and L.brevis cause little if any degradation of the oxalate, even if theydisplay significant growth.

On the other hand, L. acidophilus and S. thermophilus degrade theoxalate at both concentrations, despite the reduced growth in thepresence of ammonium oxalate 20 mM. B. infantis, finally, exhibits verygood degradation activity and is not inhibited by the oxalate at eitherconcentration.

TABLE 1 10 mM 20 mM Microbial content Microbial content Degra- (CFU/ml ×10⁶) Degra- (CFU/ml × 10⁶) dation Final dation Final Oxalic acid (%) t₀growth (%) t₀ growth L. brevis 0.94 27 130 0.73 27 120 L. acidophilus11.79 25 130 3.41 25 52 L. plantarum 1.42 32 270 0.00 32 230 S.thermophilus 2.31 2.5 26 3.06 2.5 9 B. infantis 5.26 36 300 2.18 36 230

EXAMPLE 2 Cases

After informed consent, 7 patients with hyperoxaluria were administeredStreptococcus thermophilus orally 6 g per day for 3 weeks, and oxaluriawas determined in 24 hours at time 0 and after 3 weeks.

The oxalate in urine was determined by standard methods employed inclinical chemistry and expressed as mg of oxalate in urine collected ina period of time of 24 hours.

After 3 weeks, the oxalaemia results were as follows:

Patient Before After 1 50 7 2 47 30 3 41 31 4 95 56 5 62 14 6 58 34 7 4912

It is clear that in patients with high levels of oxalate in the urine,the treatment brought about a notable reduction of the oxalate levels.

1. A method for the prophylaxis or treatment of hyeroxaluria and thedisorders associated therewith which comprises administrating to ananimal in need thereof from 0.5 to 4 g/day of a bacterial combinationcomposition comprising: (a) Streptococcus thermophilus in admixture with(b) at least one bacterial strain selected from the group consisting ofLactobacillus brevis, Lactobacillus acidophilus, Lactobacillusplantarum, Bifidobacterium infantis, Bifidobacterium longum andBifidobacterium breve or mixtures thereof.
 2. The method of claim 1,wherein the strain of Lactobacillus brevis is DSM 11988 or mutants andderivatives thereof.
 3. The method of claim 1, wherein the ratio of theconcentration of Bifidobacterium infantis, Bifidobacterium longum andBifidobacterium breve in the mixture, expressed in CFU/g of composition,are 1:1:1.
 4. The method of claim 1, wherein the ratio of theconcentrations of Streptococcus thermophilus and the Lactobacillusstrain, expressed in CFU/g of composition, is from 1000:1 to 1:1000. 5.The method of claim 1, wherein the ratio of the concentrations ofStreptococcus thermophilus and the Bifidobacterium strain, expressed inCFU/g of composition, is from 1000:1 to 1:1000.
 6. A method for theprophylaxis or treatment of hyperoxaluria and the disorders associatedtherewith comprises administrating to a subject in need thereof from 0.5to 4 g/day of a bacterial combination composition comprising: (a)Streptococcus thermophilus in admixture with (b) at least one bacterialstrain selected from the group consisting of Lactobacillus brevis,Lactobacillus acidophilus, Lactobacillus plantarum, Bifidobacteriuminfantis, Bifidobacterium longum and Bifidobacterium breve or mixturesthereof.
 7. The method of claim 6, wherein the subject is a human.
 8. Adietary or pharmaceutical composition comprising at least one strain ofa bacteria selected from the group consisting of Streptococcusthermophilis, Lactobacillus brevis, Lactobacillus acidophilus,Lactobacillus plantarum, Bifidobacterium infantis, Bifidobacteriumlongum and Bifidobacterium breve in combination with ahyperoxaluria-inducing drug.
 9. The composition of claim 8, in which thedrug is orlistat.